Riboswitches are dynamic RNA machines that detect and respond to metabolites, second messengers and ions. In most cases, binding invokes a conformational change in the ligand binding, or aptamer, domain that interfaces with the expression platform to regulate production of the downstream genes. This proposal explores five riboswitch systems including: the second-messenger-signaling riboswitches that respond to c-di-GMP, c-di-AMP and c-AMP-GMP, the dual-aptamer glycine riboswitch that regulates genes involved in glycine metabolism, and the pfl riboswitch that regulates genes involved in purine biosynthesis and the folate cycle. This project will undertake structural, chemical and genetic approaches to understand these RNA machines, their aptamer domains, their small molecule effectors, and the genes whose expression they regulate. The cyclic dinucleotides c-di-GMP, c-di-AMP and c-AMP-GMP control medically important phenotypes, including biofilm formation, sporulation, motility and virulence factor production. c-di-GMP analogues have been identified that bind their riboswitch targets but are resistant to nuclease degradation. Phenotypic effects of treating bacteria with these second messenger mimics will be explored and the cellular uptake properties of the analogues improved. The biochemistry and structure of the recently discovered c-di-AMP riboswitch in complex with its dinucleotide effector will be explored and analogues developed to target this signaling system. Similar work will be done on the c-AMP-GMP riboswitch using studies of the c-di-GMP riboswitch as a starting point. The glycine riboswitches control genes responsible for amino acid homeostasis. They have a complex dual aptamer architecture with a single downstream expression platform. The molecular circuitry and regulatory wiring of this dual aptamer riboswitch will be investigated. The pfl riboswitch commonly regulates expression of enzymes involved in the folate cycle. It is controlled by the purine biosynthetic intermediate 5-aminoimidazole 4-carboxamide monophosphate (ZMP or AICAR), which may serve as an alarmone in bacteria and is a signaling molecule in humans. The project aims to determine the X-ray crystal structure of the RNA aptamer-ligand complex and biochemically characterize the kinetics, thermodynamics and specificity of the interaction. In addition to the riboswitch work, three new classes of catalytic RNAs have been discovered and the structure of the first, the Twister ribozyme, has been determined through the ongoing work of this program project. Nucleotide Analog Interference Mapping will be used to explore the chemical basis of catalysis by these widely-distributed and previously unknown self-cleaving RNAs.